WO2006096451A2 - Anticorps de methionine sulfoxyde - Google Patents

Anticorps de methionine sulfoxyde Download PDF

Info

Publication number
WO2006096451A2
WO2006096451A2 PCT/US2006/007424 US2006007424W WO2006096451A2 WO 2006096451 A2 WO2006096451 A2 WO 2006096451A2 US 2006007424 W US2006007424 W US 2006007424W WO 2006096451 A2 WO2006096451 A2 WO 2006096451A2
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
meto
antibodies
oxidized
proteins
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2006/007424
Other languages
English (en)
Other versions
WO2006096451A3 (fr
Inventor
Jackob Moskovitz
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Kansas
Original Assignee
University of Kansas
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of Kansas filed Critical University of Kansas
Publication of WO2006096451A2 publication Critical patent/WO2006096451A2/fr
Publication of WO2006096451A3 publication Critical patent/WO2006096451A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/16—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from plants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere

Definitions

  • ROS reactive oxygen species
  • Methionine oxidation is one of the most common posttranslational modifications to proteins mediated by reactive oxygen species (ROS) that may alter protein structure/function.
  • ROS reactive oxygen species
  • Oxidative damage to proteins is considered to be one of the major causes of aging and age-related diseases, and thus mechanisms have evolved to prevent or reverse these modifications.
  • Pathology analysis performed on postmortem Alzheimer's diseased brains revealed higher levels of MetO-protein (Dong 2003) and carbonyl groups that correlated with diminished Msr activity in comparison to control brains (Gabbita 1999).
  • MetO targeted proteins will greatly enhance the knowledge about processes leading to cellular malfunction associated with protein damage, thereby providing information that could be pivotal in developing of novel therapeutics for treating oxidative stress-associated diseases.
  • the proteins include calmodulin (Gao 1998), Ikappa B (Kanayama 2002, Mohri 2002), and the voltage-dependent K (+) channel (Shaker) (Giorba 1997).
  • the current knowledge about the identity of methionine-oxidized proteins, either in their fully damaged state or their intermediate functional stage in vivo, is still very limited.
  • the present invention is directed to the production of antibodies specific for MetO-proteins. It is anticipated that the development of a new method for MetO-protein analysis will greatly advance research involving post-transnational modification to proteins. More specifically, identification of MetO-proteins in physiological processes that are affected by ROS production (e.g. aging and neurodegenerative diseases) will shed light on cellular processes that can become toxic due to the accumulation of specific methionine-oxidized proteins.
  • ROS production e.g. aging and neurodegenerative diseases
  • the present invention is directed to novel antibodies that recognize MetO amino acid residues in proteins.
  • the present invention is directed to the method of making antibodies specific for MetO proteins.
  • the present invention is directed to the preparation of antibodies specific for MetO by using an antigen comprised oxidized zein proteins.
  • the oxidized modified recombinant protein of the Zea mays high sulfur Zein gene (18kDa protein (DZS 18)) is used as the antigen.
  • the antigen used to generate the antibodies perferably is comprised of at least 23% methionine.
  • the present invention is directed ot a method of making the antidivies comprising (a) oxidizing a zein protein to provide a methionine sulfoxide containing zein protein; (b) immunizing an animal with the oxidized zein protein or an immunogenic fragment thereof, under conditions to elicit an antibody response; and (c) isolating antibody producing cells from the animal.
  • Suitable oxidizing agents are well known in the art, and a preferred oxidizing agent is hydrogen peroxide.
  • the antibody producing cells are fused with immortalized cells to form monoclonal antibody-producing hybridoma cells.
  • the antibodies of the present invention are specific for the MetO residues in proteins such as glutamine synthestase, oxidized glyceraldehyde 3 -phosphate dehydrogenase, oxidized calmodulin, lkappa B, and voltage-dependent K(+) channel.
  • the present invention is directed to a method for identifmg proteins that exhibit increased MetO level with oxidative stress conditions, slenium deficiency, age, age and/or neurodegenerative-associated diseases, or a combination of the above.
  • the present invention is directed to a kit comprising the isolated antibody for screening for expression of a polypeptide containing methionine sulfoxide.
  • the present invention includes a diagnostic test for a condition or disease associated with the expression of methionine sulfoxide containing polypeptides in a biological sample.
  • the method includes combining the biological sample with the antibodies of the invention, under conditions suitable for the antibody to bind the polypeptide and form an antibody: polypeptide complex, and detecting the complex, wherein the presence of the complex correlates with the presence of the polypeptide in the biological sample.
  • the present invention be limited to antibodies specific for MetO-proteins from certain animals.
  • the antibody is specific for rabbit MetO-proteins (e.g. glyceraldehydes 3-phosphate dehydrogenase, GAPD).
  • the antibody is specific for human, rodents, primates, yeast, plant, fungi, or bacterial MetO-proteins. That is, the antibody may be used with biological extracts of all kinds containing MetO-proteins.
  • the present invention be limited to antibodies generated in a particular animal.
  • a variety of animals are useful for generating the antibodies of the present invention, hi one embodiment, the antibody is generated in an animal selected from a mouse, a rat, a horse, a goat, a chicken, and a rabbit, hi some embodiments, the antibodies are collected from the blood of the animal.
  • the present invention be limited to the nature of the antibodies, as a variety of antibody types are contemplated.
  • the antibodies are monoclonal.
  • the antibodies are humanized.
  • the antibodies are chimeric.
  • the antibodies are polyclonal. Additional aspects of the invention, together with the advantages and novel features appurtenant thereto, will be set forth in part in the description that follows, and in part will become apparent to those skilled in the art upon examination of the following, or may be learned from the practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims. Brief Description of the Drawings
  • FIG. 1 is a western blot analysis of oxidized and non-oxidized proteins using primary antibodies against MetO-DZS18.
  • Lane 18 shows the non-oxidized DZS 18 protein.
  • Lane 0x18 shows the oxidized DZSl 8.
  • Lane GS shows the native glutamine synthetase (GS) (E. CoIi).
  • Lane OxGS shows oxidized GS.
  • Lane GAPD shows native glyceraldehyde 3- phosphate dehydrogenase (Rabbit).
  • Lane OxGAPD shows oxidized GAPD. Five microgram of each protein was subjected to the 4-20% SDS-gel electrophoresis. Oxidation of the proteins was performed using 0.3% of hydrogen peroxide for 2 hours at 37 °C. Molecular mass of proteins was as follows: 18: 18kDa; GS: 5OkDa; GAPD: 36kDa.
  • FIG. 2 shows the reaction of anti-MetO-DZS18 with proteins of several mouse tissues with and without exposure to hydrogen peroxide.
  • Protein extracts from several mouse tissues were treated with and without 10OmM hydrogen peroxide.
  • SDS-protein-gel- electrophoresis (4-20%) of the proteins the MetO-proteins were detected in a western blot analysis using the anti-MetO-DZS18 antibodies.
  • kDa indicates masses of the various protein molecular markers.
  • FIG. 3 shows the detection of MetO-proteins in yeast strains in vivo by using the anti-MetO-DZS18 antibodies.
  • the yeast strains: OP (MsrA overproducing strain) and MT (null mutant msrA strain) were grown in the presence or absence of ImM hydrogen peroxide until their growth rate reached 150 klett units. Following their growth, the cells were harvested, extensively washed with PBS, and disrupted in the presence of PBS and proteases inhibitors cocktail (Roche). Thirty micrograms of protein extracts from each strain were subjected to 2% SDS-protein-gel-electrophoresis followed by western blot analysis using the anti-MetO-DZS18 antibodies.
  • FIG. 4 shows the detection of MetO-proteins in human B-cell lymphocytes in vivo by using the anti-MetO-DZS18 antibodies.
  • Human B-cell lymphocyte cell lines were grown in the absence or presence of elevated concentrations of hydrogen peroxide for about two hours at about 37 0 C. Following the incubations the cells were extensively washed with PBS and then disrupted in PBS in the presence of proteases inhibitors cocktail (Roche).
  • FIG. 5 shows the MetO-protein levels in tissues of wild type and MsrA ' / ⁇ mouse strains, fed with SD diet. Black or hatched bars represent wild type or MsrA/ ' mice, respectively.
  • "Cer” refers to the brain cerebrum; "Ceb” to the brain cerebellum; "Lun” to the lungs; and "Hrt” to the heart. Analysis for MetO-protein was performed using amino acid analysis, according to previously described method (Reddy 1994). Five animals were used for each averaged data point Detailed Description of Preferred Embodiment
  • isolated means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally occurring antibody present in a living animal is not isolated, but the same antibody, separated from some or all of the coexisting materials in the natural system, is isolated.
  • antibody refers to intact molecules as well as fragments thereof, such as Fab, F(ab') 2 , and Fv, which are capable of binding the epitopic determinant. Methods of making these fragments are known in the art. (See for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1988), incorporated herein by reference).
  • antibody includes monoclonal antibodies, polyclonal antibodies, chimeric, or humanized antibodies.
  • the antibodies of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2), or subclass of immunoglobulin molecule.
  • the immunoglobulin molecules of the invention are IgG.
  • humanized antibody refers to antibody molecules in which amino acids have been replaced in the non-antigen binding regions in order to more closely resemble a human antibody, while still retaining the original binding ability.
  • the antibodies of the present invention may be generated by any suitable method known in the art.
  • Polyclonal antibodies to the antigen of interest can be produced by various procedures well known in the art.
  • the oxidized zein proteins containing numerous MetO residues can be administered to various host animals including, but not limited to, rabbits, mice, rats, etc. to induce the production of sera containing polyclonal antibodies specific for the antigen.
  • adjuvants may be used to increase the immunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum. Such adjuvants are also well known in the art.
  • Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.
  • monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies and T-CeIl Hybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporated by reference in their entireties).
  • the term “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology.
  • the term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.
  • mice or rabbits can be immunized with the MetO-containing zein proteins of the present invention.
  • an immune response e.g., antibodies specific for the antigen are detected in the mouse serum
  • the mouse spleen is harvested and splenocytes isolated.
  • the splenocytes are then fused by well-known techniques to any suitable myeloma cells, for example, cells from cell line SP20 available from the ATCC.
  • Hybridomas are selected and cloned by limited dilution.
  • hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding a polypeptide of the invention.
  • Ascites fluid which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.
  • the present invention provides methods of generating monoclonal antibodies as well as antibodies produced by the method comprising culturing a hybridoma cell secreting an antibody of the invention wherein, preferably, the hybridoma is generated by fusing splenocytes isolated from a mouse immunized with an antigen of the invention with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete an antibody able to bind a polypeptide of the invention.
  • polyclonal or monoclonal antibodies can be further purified, for example, by binding to and elution from a matrix to which the polypeptide or a peptide to which the antibodies were raised is bound.
  • a matrix to which the polypeptide or a peptide to which the antibodies were raised is bound.
  • Those of skill in the art will know of various techniques common in the immunology arts for purification and/or concentration of polyclonal antibodies, as well as monoclonal antibodies (See for example, Coligan, et al., Unit 9, Current Protocols in Immunology, Wiley Interscience, 1994, incorporated by reference).
  • the term "specific for" when used in reference to the interaction of an antibody protein or peptide means that the interaction is dependent upon the presence of a particular structure (i.e., the antigenic determinant or epitope) on the protein; in other words the antibody is recognizing and binding to a specific protein structure rather than to proteins in general (i.e. non-specific or background binding).
  • Zeins The endosperm of maize (Lea mays) seeds contains several classes of alcohol- soluble storage proteins called zeins, which together make up nearly 50% of the total seed protein content. Some Zeins proteins are rich in sulfur amino acids (Met and Cys). Thus, in the present invention, zeins were used in designing a high rich-MetO protein. Zeins can be separated into four major classes (as determined by their sequence and apparent molecular mass following SDS/PAGE: ⁇ (19 and 22 kDa), ⁇ (15 kDa), ⁇ (16 and 27 kDa) and ⁇ (10 kDa) Zeins.
  • a recombinant of His-tagged DZS 18 was overexpressed in E. CoIi. bacteria and affinity purified on a nickel column.
  • the methionines of the pure recombinant DZSl 8 were then oxidized by hydrogen peroxide and then injected into a rabbit to create antibodies specifically reacting against MetO-protein.
  • the 5'-forward DNA primer started at 5'end of the DZSl 8 open reading frame (starting 66bp downstream of the first ATG cogon).
  • the 3'-reverse complement DNA primer was designed to start at the un-translated area forwarding towards the DZSl 8 stop codon.
  • the 5'-forward primer contained a unique Bam HI restriction site and the 3 '-reverse complement primer contained a unique Hindlll restriction site.
  • the template was a plasmid harboring the DZS 18 gene and its upstream and downstream flanking regions (Swarup 1995) (kindly provided by Dr. J. Messing, Rutgers University, NJ).
  • Taq polymerase, NTPs, and appropriate reaction buffer were also used (Kit purchased from Roche Inc).
  • the resulting PCR product was digested with BamRI and Hindlll enzymes and sub-cloned into a pQE30 vector (Qiagen) at the complementary restriction sites.
  • the overexpressed protein was then subjected to a nickel column and purified to homogeneity according to the manufacture procedure (BD Biosciences). The purified recombinant protein was oxidized in the presence of 0.3% hydrogen peroxide diluted in water at 37 0 C for 2 hours.
  • the protein was oxidized with 0.3% hydrogen peroxide for about hours at about 37 °C.
  • the oxidized protein (MetO-DZS18) was dialyzed against PBS, pH 7.4, to remove remaining oxidant and subjected to amino acid analysis, as previously described (Reddy 1994).
  • the protein was found to be fully oxidized in its methionine moiety.
  • the confirmed methionine oxidized DZS 18 was injected into a rabbit in multiple injections of lOO ⁇ g in 2 weeks intervals to produce anti-MetO-DZS18 antibodies.
  • the rabbit was bled following the second injection in between the injections and the titer and specificity of the antibodies was tested by western blot analysis using MetO-DZS18 as a probe as discussed in Example 2.
  • Example 2 Antibodies Produced against Meto-DZs318 Show Specificity Towards Proteins Containing MetO
  • Example 1 it was demonstrated that the antibodies from Example 1 showed specificity towards the MetO-DZS18 protein and did not react with the non-oxidized form of the protein (DZSl 8).
  • the antibodies reacted with several non-homologus proteins in their oxidized forms: rabid glutamine synthetase (GS) (Sigma) and E. coli glyceraldehyde 3-phosphate dehydrogenase (GAPD) (provided by B. Berlett, NIH). This suggests specific reactivity of the antibodies towards methionine sulfoxide residues. All the methionines in the oxidized proteins were found to be oxidized, as judged by amino acid analysis.
  • the MetO-DNZ18 migrates as a 36-kDa protein due to the mass contribution by the addition of 6-Histidine tag to its N-terminus and oxygen to all of its methionines.
  • the native GS shows reaction with antibodies (FIG. 1) as it is naturally oxidized under its recommended storage conditions at 4 0 C.
  • the oxidized GS (OxGS) reacted slightly stronger with the antibodies and migrated slower in the gel due its enhanced methionine oxidation.
  • the reaction intensity of the antibodies with the proteins generally correlates with the percent of the methionine residues in each tested protein (OxI 8, 25%; OxGs, 3.6%; and OxGAPD, 3.0%), suggesting full methionine oxidation in all proteins.
  • the present invention contemplates testing of several other proteins by western blot analysis to evaluate the antibodies binding specificity to pure MetO-proteins. For example, aprotinine (Sigma) (one methionine that is surface exposed), BSA (Sigma), calmodulin (sigma), and Ikappa B will be tested. The latter two proteins contain methionines that their oxidation have shown to alter their function.
  • yeast strains were grown in the presence of absence of 1 mM hydrogen peroxide.
  • One strain was enriched in its MetO-reduction ability (OP, an overproducing strain of MsrA) and a one strain was compromised its MetO-reduction ability (MT, a null mutant strain of msrA: H9strain, Mata ura3-52 his 5 Ieu2).
  • the cells were grown until their growth reached 150 klett units. Following their growth, the cells were harvested, extensively washed with PBS, and disrupted in the presence of PBS and proteases inhibitors cocktail (Roche).
  • the strain (MT) that was more sensitive to methionine oxidation produced a dominant protein band following reaction with the anti- MetO-DZS18 antibodies even without the hydrogen peroxide treatment. Moreover, the reaction with this protein band that was enhanced following exposure of the cells to hydrogen peroxide. Accordingly, the resistant strain to methionine oxidation (OP) showed no detectable reactions with the anti-MetO-DZS18 antibodies (FIG. 3). The specificity of the antibodies to single MetO residues was confirmed by the fact that when free MetO was co-incubated with the anti-MetO-DZS18 antibodies (during the western blot analysis) the reactivity of the protein band with the antibodies was diminished (FIG.
  • the additional presented data provides supportive evidence for the concept that indeed the anti-MetO-DZS18 antibodies are reacting specifically towards multiple and single MetO residues.
  • the average percent of total methionine residues in a protein is only about 2%.
  • the percentage of the surface exposed methionine is even lower than 2% as methionine by its nature is a hydrophobic amino acid. Therefore, most of the methionine residues are predicted to be buried within the molecular structure of the protein; thereby less excessive to oxidation by most oxidants.
  • a combination of two steps will be performed: one is to fractionate and enrich the extracted proteins according to their molecular mass (i.e to low, medium, and high molecular mass fractions) using an HPLC sizing column techniques; and one is to apply 2D- gel-electrophoresis technique to have a better separation and resolution of the proteins. Combining the two steps is expected to enhance the level of detection of low abundant MetO- proteins.
  • the proteins can be separated according to their molecular mass by a sizing column (Bio-Rad) using a common HPLC procedure. Eluted proteins will be collected and pooled into three groups of low (5-40 kDa), medium (40-8OkDa), and high (80- 20OkDa) molecular mass proteins. Furthermore, each group of proteins will be also separated into additional two groups: one group of proteins that will bind to a Mono-Q column (Pharmacia) in the presence of 5OmM Tris at pH, 7.4 and to one group of proteins that does not bind, respectively. Based on these protein size and charge separations, it is expected that the relative levels of under-representative MetO-proteins will be elevated in all or some of the six groups of proteins relative to their levels in crude extract.
  • a sizing column Bio-Rad
  • Eluted proteins will be collected and pooled into three groups of low (5-40 kDa), medium (40-8OkDa), and high (80- 20OkDa
  • human B-lymphocytes cell line was used as a model for MetO-accumulation in human cell culture.
  • the B-lymphocytes cell line was exposed to increasing concentrations of hydrogen peroxide and protein extracts were made following incubation for 2 hours at 37 0 C.
  • Western blot analysis performed on corresponding protein extracts revealed that even the absence of hydrogen peroxide there were proteins that were recognized to have MetO residues (FIG 4).
  • FIG. 4 Western blot analysis
  • FIG. 4 Western blot analysis performed on corresponding protein extracts revealed that even the absence of hydrogen peroxide there were proteins that were recognized to have MetO residues.
  • FIG. 4 Western blot analysis
  • Enrichment and purification of MetO-proteins from biological extracts may be achieved by using a MetO-affinity column that is based on the specific anti-MetO-DZS18 antibodies.
  • the procedure that covalently binds the antibodies to the column and its use for binding and isolating of the targeted proteins will be performed as follows: 0.1 ml of Protein- A coupled matrix (Pierce) will be washed with deionized distilled water (ddW) then with 50 mM sodium borate buffer, pH 8.2. Excess amount of the buffer will be removed.
  • Anti-MetO antibodies (2mg/ml) will be diluted in 0.1 ml of antibody binding buffer (50 mM sodium borate buffer, pH 8.2) so that the final concentration of antibody will be 0.15 mg/ml.
  • the diluted antibody solution will be added into protein-A conjugated agarose beads and incubated for 30 minutes with gentle mixing. Alternatively, overnight incubation at 4 0 C will be employed. Unbound flow through antibody solution will be collected from the column and the gel will be washed five times with 0.1 ml of antibody binding buffer followed by washing with 0.1 ml of cross-linking buffer (0.2M triethanolamine, pH 8.2).
  • DSS disuccycinilamide subarate
  • the gel will be washed extensively as follows: (1) Five times with 0.1 ml of ddW; (2) Two times with 0.1 ml of 1.0 M NaCl; (3) Two times with 0.1 ml of 0.1 M Glycine, pH 2.8; and (4) Three times with 0.1 ml of ddW.
  • the anti-MetO Antibody cross-linked agarose beads will be kept in PBS, pH7.4, containing 0.02% sodium azide at 4 °C until use.
  • the binding capability of the column to pure proteins will be tested using several proteins, such as rabbit GAPD, E. coli GS, DZS 18, aprotinine, BSA, Calmodulin, Ikappa B, and beta-amyloid (AnaSpec, CA) (lOO ⁇ g each) at their normal or oxidized form. Proteins will be oxidized by 0.3% of hydrogen peroxide for 2 hours at 37 °C and then will be dialyzed against PBS, pH 7.4.
  • Both the oxidized and non-oxidized proteins will be subjected to separated columns, washed with PBS, and eluted with 0.1 M Glycine, pH 2.8.
  • the eluted proteins will be analyzed for their MetO content by amino acid analysis, as previously described (Reddy 1994). It is expected that the oxidized proteins will bind to the columns much more efficiently than their non-oxidized forms, respectively.
  • FIG. 1 showed that GS and GAPD cross-reacted with the antibodies suggesting that even a single MetO residue is being recognized by the antibodies (as both proteins have multiple methionine residues that are located apart from each other).
  • free MetO was able to compete with the antibodies reactions suggesting that at least some of the antibodies can recognize a single MetO residue (FIG. 3).
  • the titer of the specific antibodies raised against single MetO residue may be limited. To overcome this possible limitation and enrich the titer of these antibodies, the antibodies will be affinity purified on a resin that is covalently bound to the N-terminus of the amino acid MetO.
  • the Methionine-resin compound will be purchased from Sigma and swollen in 1 ml of ddW.
  • the MetO-resin will be oxidized with 0.3% of hydrogen peroxide for 2 hours at 37 0 C, for making the MetO-resin.
  • the column will be washed 5 times with 2 ml of PBS.
  • 2ml of the antibodies will be subjected to the column at slow rate (O.lml/min) followed by multiple washing steps (5 washes with 2ml PBS). Elution of the bound antibodies will be carried out by applying total of 5 ml of 0.1M glycine, pH 2.8 in aliquots of ImI.
  • the present invention includes a diagnostic test for a condition or disease associated with the expression of methionine sulfoxide containing polypeptides in a biological sample.
  • the method includes combining the biological sample with the antibodies of the invention, under conditions suitable for the antibody to bind the polypeptide and form an antibody :polypeptide complex, and detecting the complex, wherein the presence of the complex correlates with the presence of the polypeptide in the biological sample.
  • Biological extracts of yeast and mice tissues will be screened for the existent of MetO-protein.
  • msrA null mutant yeast strain will be grown with and without the presence of 2 mm hydrogen peroxide in the growth media, as previously described (Moskovitz 1997, Moskovitz 2).
  • Cell extracts will be made at early growth rate in PBS buffer, pH 7.4, in the presence of protease inhibitors cocktail (Roche).
  • Equal amounts of proteins from extracts (about 1 Omg), made from cell that were and were not exposed to hydrogen peroxide, will be subjected to two separated anti-MetO columns according to the procedure described herein. Proteins eluted from each column will be analyzed for total MetO-protein content by amino acid analysis (Reddy 1994).
  • protein extracts will be divided into three enriched groups of molecular mass proteins: low, medium, and high, using HPLC-sizing and Mono-Q columns, as described in above. Then, the proteins will be separated using 2D-protein-gel- electrophoresis followed by western blot analysis probed with the anti-MetO-DZS18 antibodies. Proteins that will be detected by the antibodies will be extracted from the gel for final identification. In both methods, their identities will be determined by applying LC/MS and common mass spectrometry techniques. Briefly, each purified protein will be subjected to a tryptic digestion. The combination of the molecular masses of the resulting peptides (as determined by mass spectrometry) will provide the digestion pattern information need for the protein identification (using the protein data bank).
  • mice that were treated with hydrogen peroxide will have much higher oxidized proteins than the non-treated cells and may have different MetO-protein profile, respectively.
  • SD selenium deficient mice
  • each crude extract will be separated either by size or charge based column while fractionating the eluted proteins into series of collecting tubes.
  • each fraction will be subjected to either ID or 2D -protein-gel-electrophoresis followed by western blot analysis using the anti-MetO-DZS18 antibodies.
  • the identity of each potential MetO-protein will be determined by mass- spectrometry techniques.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Hematology (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Urology & Nephrology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Microbiology (AREA)
  • Analytical Chemistry (AREA)
  • Cell Biology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Botany (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne des anticorps spécifiques aux restes de méthionine sulfoxyde sur des protéines. Les anticorps sont préparés au moyen de protéines de zéine riches en méthionine, qui sont oxydées, utilisées comme antigènes.
PCT/US2006/007424 2005-03-08 2006-03-02 Anticorps de methionine sulfoxyde Ceased WO2006096451A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US65964905P 2005-03-08 2005-03-08
US60/659,649 2005-03-08

Publications (2)

Publication Number Publication Date
WO2006096451A2 true WO2006096451A2 (fr) 2006-09-14
WO2006096451A3 WO2006096451A3 (fr) 2007-07-12

Family

ID=36953857

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2006/007424 Ceased WO2006096451A2 (fr) 2005-03-08 2006-03-02 Anticorps de methionine sulfoxyde

Country Status (2)

Country Link
US (2) US20060204494A1 (fr)
WO (1) WO2006096451A2 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023081825A2 (fr) * 2021-11-05 2023-05-11 University Of Kansas Immunisation active contre les maladies associées aux amyloïdes et au vieillissement

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4281061A (en) * 1979-07-27 1981-07-28 Syva Company Double antibody for enhanced sensitivity in immunoassay
US5834278A (en) * 1996-05-01 1998-11-10 The Rockefeller University Bacterial peptide methionine sulfoxide reductase an adhesion-associated protein, and antibiotic therapies based thereon
WO2001049852A1 (fr) * 2000-01-05 2001-07-12 The Regents Of The University Of California Mais transgenique contenant des genes de pbf recombines
US20050160488A1 (en) * 2000-11-07 2005-07-21 Pioneer Hi-Bred International, Inc. Grain quality through altered expression of seed proteins
US20030219734A1 (en) * 2001-04-13 2003-11-27 Biosite Incorporated Polypeptides related to natriuretic peptides and methods of their identification and use
US20030134339A1 (en) * 2002-01-14 2003-07-17 Thomas Brown Proteomics based method for toxicology testing

Also Published As

Publication number Publication date
US20060204494A1 (en) 2006-09-14
US8409824B2 (en) 2013-04-02
WO2006096451A3 (fr) 2007-07-12
US20100068205A1 (en) 2010-03-18

Similar Documents

Publication Publication Date Title
Huang et al. Zeta-crystallin, a novel lens protein from the guinea pig
Mattsson et al. Molecular and immunological characterization of Can f 4: a dog dander allergen cross‐reactive with a 23 kDa odorant‐binding protein in cow dander
Bromilow et al. Comprehensive proteomic profiling of wheat gluten using a combination of data-independent and data-dependent acquisition
Chassaigne et al. Resolution and identification of major peanut allergens using a combination of fluorescence two-dimensional differential gel electrophoresis, Western blotting and Q-TOF mass spectrometry
Oien et al. Detection of oxidized methionine in selected proteins, cellular extracts and blood serums by novel anti-methionine sulfoxide antibodies
Neri-Castro et al. Detection and quantification of a β-neurotoxin (crotoxin homologs) in the venom of the rattlesnakes Crotalus simus, C. culminatus and C. tzabcan from Mexico
Recuenco-Muñoz et al. Targeted quantitative analysis of a diurnal RuBisCO subunit expression and translation profile in Chlamydomonas reinhardtii introducing a novel Mass Western approach
US6953666B1 (en) Biomarkers for oxidative stress
Nikolić et al. Employment of proteomic and immunological based methods for the identification of catalase as novel allergen from banana
Hanson et al. A receptor for the import of proteins into human mitochondria
CN112567039A (zh) 阿尔茨海默氏病的诊断药物和诊断方法
US5405749A (en) Method for identifying and purifying a cancer associated retinopathy autoantigen, and testing patient serum for the autoantibody to the autoantigen
EP3149193A1 (fr) Dosage de complexe de facteur antisécrétoire
US8409824B2 (en) Methionine sulfoxide antibodies
CA2348685A1 (fr) Biomarqueurs du stress d'oxydation
EP1779115B1 (fr) Procédé de dosage du gluten
Singh et al. A new candidate protein for high lysine content in wheat grain
Guo et al. Proteomic analysis of peanut seed storage proteins and genetic variation in a potential peanut allergen
Wang et al. PPIase is associated with the diversity of conotoxins from cone snail venom glands
US5753522A (en) Purified protein for identifying a cancer-associated retinopathy autoantibody
Perez-Gregorio et al. Chromatographic and mass spectrometry analysis of wheat flour prolamins, the causative compounds of celiac disease
Zhang et al. High-specificity quantification method for almond-by-products, based on differential proteomic analysis
Johansson et al. Identification of flotillin-1 as an interacting protein for antisecretory factor
Hirohashi et al. Molecular cloning and characterization of maize Toc34, a regulatory component of the protein import machinery of chloroplast
CN119039446B (zh) 抗gad65抗体及其应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: DE

NENP Non-entry into the national phase

Ref country code: RU

122 Ep: pct application non-entry in european phase

Ref document number: 06736699

Country of ref document: EP

Kind code of ref document: A2